Periplasmic methionine sulfoxide reductase (MsrP)—a secondary factor in stress survival and virulence of Salmonella Typhimurium

Abstract Among others, methionine residues are highly susceptible to host-generated oxidants. Repair of oxidized methionine (Met-SO) residues to methionine (Met) by methionine sulfoxide reductases (Msrs) play a chief role in stress survival of bacterial pathogens, including Salmonella Typhimurium. Periplasmic proteins, involved in many important cellular functions, are highly susceptible to host-generated oxidants. According to location in cell, two types of Msrs, cytoplasmic and periplasmic are present in S. Typhimurium. Owing to its localization, periplasmic Msr (MsrP) might play a crucial role in defending the host-generated oxidants. Here, we have assessed the role of MsrP in combating oxidative stress and colonization of S. Typhimurium. ΔmsrP (mutant strain) grew normally in in-vitro media. In comparison to S. Typhimurium (wild type), mutant strain showed mild hypersensitivity to HOCl and chloramine-T (ChT). Following exposure to HOCl, mutant strain showed almost similar protein carbonyl levels (a marker of protein oxidation) as compared to S. Typhimurium strain. Additionally, ΔmsrP strain showed higher susceptibility to neutrophils than the parent strain. Further, the mutant strain showed very mild defects in survival in mice spleen and liver as compared to wild-type strain. In a nutshell, our results indicate that MsrP plays only a secondary role in combating oxidative stress and colonization of S. Typhimurium.


Introduction
Typhoidal and non-typhoidal Salmonella (NTS) are the major etiological agents of gastro-intestinal diseases in humans.Salmonella is associated with more than 100 million annual cases of human infections worldwide (Sears et al. 2021 ).Typhoidal Salmonella is gener all y r estricted to human and include ser ov ars like S .enterica Typhi and S .enterica P ar atyphi, whic h cause enteric fe v er (Bula-Rudas et al. 2015 ).NTS includes S. enterica ser ov ar Typhim urium ( S. Typhimurium) and S. enterica serovar Enteritidis ( S .Enteritidis) (Gal-Mor et al. 2014 ).NTS-associated infection is common in old and imm unocompr omised individuals (Gordon 2008 ) and is estimated to cause over 90 million cases and 100 000 annual deaths w orldwide (Majo wicz et al. 2010 ).Inter estingl y, S .Typhim urium accounts for about 21.9% of cases of poultry salmonellosis in India (Kumar et al. 2019 ).Most of the infected poultry birds serve as asymptomatic reservoirs of NTS.Howe v er, poultry pr oducts ar e one of the major sources of NTS infection in humans (Ferrari et al. 2019 ).
Inside the host, S .Typhimurium experiences several stresses, including extreme pH of gastric and intestinal secretions, antimicrobial peptides, and various reactive oxygen, chlorine, and nitrogen species (ROS , RCS , and RNS).Salmonella , being an intr acellular pathogen, can surviv e and r eplicate inside pha go-c ytic cells.Phagoc ytes gener ate v arious ROS , RCS , and RNS which can damage bacterial biomolecules, including DNA, proteins, and lipids (Mastroeni et al. 2000 ).RCS molecules like HOCl ar e gener ated during r espir atory burst and ar e highl y r eactive with methionine residues.To survive in such adverse envir onment of pha gocytic cells, Salmonella has e volv ed a wide range of strategies.SPI-2 encodes an effector protein SpiC, which pr e v ents the assembl y of pha gosomal oxidase and impr ov es the survival of S .Typhimurium in Salmonella -containing vacuoles (SCV) (Vazquez-Torres and Fang 2001 ).Primary antioxidant enzymes lik e catalases, supero xide dismutases, and alkyl hydr oper oxides catal yticall y act on the host-gener ated o xidants like H 2 O 2 , O 2 r − , ONOO r , r espectiv el y (Hébr ard et al. 2009, Kröger et al. 2013 ).
During r espir atory burst, the oxidants gener ated by imm une cells exceed the scav enging ca pacities of primary anti-oxidant enzymes, whic h r esults in o xidati v e modifications of differ ent biomolecules .P ossessing electron rich sulphur, cysteine, and methionine residues are highly susceptible to oxidation (Bin et al. 2017 ).After oxidation, methionine (Met) residues convert into methionine sulfoxide (Met-SO) (either R -or S -form) (Stadtman et al. 2005 ).Oxidation of Met residues can alter protein structur e, function, and consequentl y affect cellular viability.Ho w e v er, methionine sulfoxide reductases (Msrs), with the help of thioredo xin and thioredo xin r eductase can r educe Met-SO to Met and thus impr ov e the bacterial surviv al under o xidati v e str ess conditions (Zhang and Weissbach 2008 ).
Based on location, stereospecificity, and specificity to peptide bound or free Met-SO, four cytoplasmic Msrs have been identified in S .Typhim urium, namel y MsrA, MsrB, MsrC, and BisC.MsrA is specific for S -form of Met-SO and can act on both free or peptidebound Met-SO residues (Denkel et al. 2011 ).MsrB is highly specific for R -form of Met-SO and is activ e onl y on peptide-bound Met-SO residues.MsrC and BisC act on free R -and S -forms of Met-SO, r espectiv el y (Denkel et al. 2011 ).Cytoplasmic Msrs have been shown to alleviate o xidati ve stress and enhance the survival of S .Typhimurium under o xidati ve stress.msrA , msrC , and bisC m utants of S .Typhim urium wer e shown to be hypersusceptible to oxidants and phagocytic cells.Further, these strains sho w ed defective colonization in mice and poultry (Denkel et al. 2011, Denkel et al. 2013, Trivedi et al. 2015, Sarkhel et al. 2017, Nair et al. 2021 ) .
Ho w e v er, deletion of msrB gene does not affect the viability of S .Typhim urium a gainst oxidants and colonization in mice (Denkel et al. 2011 ).
Owing to its location, periplasmic compartment first comes into contact with host-generated ROS/RNS and thus Met residues in periplasmic proteins are highly vulnerable to oxidation.Therefor e, Msrs ar e r equir ed in extr acytoplasmic space to r epair Met-SO residues in oxidized proteins.Indeed, extracytoplasmic Msrs have been identified in different bacterial species, which highlights the importance of the repair of oxidized proteins in this compartment.Most of these extracytoplasmic Msrs are fusion proteins, containing MsrA and MsrB domains.PilB, a fimbrial protein in Neisseria gonorrhoeae , was the first extr acytoplasmic pr otein identified to have Msr activity (Olry et al. 2002 ).Extracytoplasmic Msrs (MsrAB) in S .pneumoniae and H . influenzae contribute to survival of these bacterial pathogens against o xidati ve stress (Saleh et al. 2013, Nasreen et al. 2020 ).
In Esc heric hia coli , unlike the abov e extr acytoplasmic Msrs, a novel periplasmic Msr (MsrP) has been identified.MsrP, along with periplasmic membrane redox protein (MsrQ) forms MsrPQ system (Gennaris et al. 2015 ).MsrP can reduce both R-as well as S -forms of l -Met-SO and have catalytic activity on both free and peptidebound l -Met-SO (Gennaris et al. 2015, Tarr a go et al. 2018 ).MsrP system has been shown to be r equir ed for survival against HOCl stress in Escherichia coli (Gennaris et al. 2015 ) and nitr osativ e str ess in Campylobacter jejuni (Hitc hcoc k et al. 2010 ).A r ecent study has identified MsrP system in S .Typhimurium which can reduce both free and peptide bound, as well as, R -and S -forms of Met-SO (Andrieu et al. 2020 ).MsrP might play a v ery important r ole in oxidativ e str ess surviv al of S .Typhim urium.Ho w e v er, the contribution of MsrP in the survival of S .Typhimurium under o xidati v e str ess and in virulence is not known.
In this study, we have assessed the contribution of MsrP under o xidati v e str ess and colonization of S. Typhim urium.

Materials and methods
All animal experiments were approved by the Institutional Animal Ethics Committee (IAEC), Indian Council of Agricultural Resear ch-Indian Veterinary Resear ch Institute (ICAR-IVRI), Izatnagar, India.All animal experimentations were performed in accordance with the ARRIVE guidelines.

Bacterial strains and plasmids
Salmonella enterica subspecies enterica ser ov ar Typhim urium str ain E-5591 ( S. Typhimurium) was procured from the National Salmonella Center , ICAR-IVRI, Izatnagar , India.The NEB-5 α strain of E. coli w as pur c hased fr om Ne w England BioLabs .T he plasmids pKD3, pKD46, and pCP20 plasmids were generously provided by Prof. Robert J. Maier, UGA, Athens, GA, USA.

Gener a tion of msrP mutant strain in S. Typhimurium
The msrP gene deletion strain ( msrP mutant strain) was constructed using lambda red recombinase-mediated gene inactivation protocol (Datsenko and Wanner 2000 ).Briefly, FRT flanked Cm cassette was amplified from pKD3 plasmid by using MsrP_New del For and MsrP_New del Rev primers (Table 1 ).The PCR conditions are detailed in Table 1 .The purified Cm cassette was transformed into lambda r ed r ecombinase expr essing S. Typhim urium.Cm-supplemented agar media was used to select recombinants and confirmed by PCR (Fig. 1 ).The Cm cassette was then r emov ed by flip recombinase (Datsenko and Wanner 2000 ).

In vitro growth evaluation
The mutation might affect in vitro proliferation of bacteria.To determine the effect of msr gene deletions on in vitro growth of S. Typhim urium, the gr owth of v arious str ains wer e assessed in LB broth or M9 media.In brief, isolated colonies of different strains wer e gr own in LB br oth or M9 media overnight at 37 • C. On the following da y, o v ernight gr own cultur es wer e diluted in 50 ml fr esh media @ 1:100 ratio and incubated in a shaker incubator at 37 • C. Aliquots of 1 ml were taken at hourly intervals and the absorbance w as recor ded at 600 nm (Nair et al. 2021 ).

HOCl and chloramine-T susceptibility assays
The susceptibility of wild-type and m utant S .Typhim urium strains to HOCl (NaOCl, Sigma) and chloramine-T (ChT) (N-Chlor o-p-toluenesulfonamide trihydr ate sodium salt, Sigma) was assessed as described earlier (Nair et al. 2021 ).In brief, the vigorousl y gr owing cultur es of wild-type and m utant str ains was pelleted, washed and suspended in PBS at an OD 600nm of 1.0.The suspensions were then treated with various concentrations of HOCl (50 and 100 μM) or ChT (100, 200, 300, 400, and 500 μM) for 30 min.After 30 min of exposure, the mix was supplemented with 10 mM (final) l -methionine and incubated for 15 min.The suspensions wer e then seriall y diluted and plated on HE a gar plates .T he plates were incubated at 37 • C and colonies were counted (Trivedi et al. 2015, Nair et al. 2021 ).

Quantification of total protein carbonyls
Total pr otein carbon yls of whole cellular pr oteome wer e determined using DNPH assays as described earlier (Apoorva et al. 2020 ).Shortly, 0 and 3 mM HOCl-exposed cultures of wild-type and m utant str ains wer e pelleted.The pellets wer e suspended in BugBuster TM Pr otein Extr action Rea gent and incubated for 30 min at r oom temper atur e, with intermittent shaking at e v ery 5 min.
Table 1.Primers used in the study and their PCR conditions.After incubation, the cell debris was r emov ed by centrifugation at 15 000 × g for 30 min at 4ºC.The supernatants were collected and incubated with 600 μl of 10 mM DNPH (in 2.5 M HCl) for an hour in dark, with intermittent vortexing at e v ery 15 min.The proteins wer e pr ecipitated by addition of 10% TCA (final) and collected by centrifugation at 15 000 × g for 20 min at 4ºC.P ellets w ere then washed twice with 10% TCA and once with ethanol and ethylacetate (1:1).The final precipitate was dissolved in 6 M guanidine hydr oc hloride and incubated for 30 min at 37ºC, with intermittent vortexing.The total protein carbonyls were determined by recording the absorbance at 355 nm using 6 M guanidine HCl as blank.Carbon yl le v els wer e calculated using the form ula: A 355 /( εl), where ε = 22 000 M −1 cm −1 (molar absorption coefficient of hydrazone is 22 000 M −1 cm −1 ), c = concentration of carbonyls in moles/litre (M), and A = absorbance at 355 nm (Reznick and P ac ker 1994 ).The total protein carbonyls were expressed as nmol/mg of proteins.

Neutrophil sensitivity assays
The sensitivity of different strains of S .Typhimurium to neutrophils was assessed as described elsewhere (Okamura andSpitznagel 1982 , Oh et al. 2008 ) with minor modifications.Briefly, goat blood (collected in EDTA) was diluted with an equal quantity of PBS.The diluted blood was then layered over a mixture of equal volumes of Histopaque 1077 and 1119 and centrifuged at 750 × g at room temperature for 45 min.Neutrophils present at the interface point between the Histopaque layers were pipetted out and washed twice with PBS by centrifuging at 250 × g for 10 min.A final wash was done with HBSS without Ca 2 + and Mg 2 + and the neutrophils were suspended in the same medium.
The vigor ousl y gr owing cultur es of v arious Salmonella str ains wer e pelleted, washed, and suspended in HBSS with Ca 2 + and Mg. 2 + Bacteria and neutrophils were mixed in a ratio of 10:1 and incubated in a CO 2 incubator at 37 • C, for 60 min.Post incubation, the mix was centrifuged at 13 000 rpm for 3 min.The supernatant w as discar ded, the pellet w as treated with 0.1% Triton X-100 (final concentration) and finally, the lysates were diluted and plated on HE agar.Plates were incubated at 37 • C and colonies were enumerated.

Competiti v e infection assays in mice
The effect of deletion of the msrP gene in the virulence of S. Typhimurium was assessed in Swiss Albino mice.
In brief, the overnight grown cultures of S .Typhimurium strain and msrP ::Cm strains were diluted in 50 ml LB broth and grown upto an OD 600nm of 0.80.The cultures were then pelleted and suspended in PBS to obtain CFUs of 4 × 10 4 /50 μl.The suspensions of two str ains wer e mixed at 1:1 ratio and a total of 100 μl containing 8 × 10 4 CFUs were inoculated in each mouse by intra-peritoneal route.Actual bacterial numbers were determined by r etr ospectiv e plating.The mice were sacrificed following 3-and 5-days post-infection and the spleen and liver were homogenized in PBS.The homogenates were diluted and plated on plain as well as antibiotic-containing agar media (HE agar for S .Typhim urium and HE a gar supplemented with Cm for msrP strain).
Competitive index (CI) was calculated as described elsewhere (Denkel et al. 2011, Kumawat et al. 2016 ).CI is the ratio of mutant to wild-type strain recovered divided by the ratio of mutant to wild-type strain inoculated.

MsrP is not essential for in vitro growth of S. Typhimurium
The growth of wild-type and mutant strains was assessed for duration of 12 hours in LB broth and M9 media.In both the media, sigmoidal-sha ped gr owth curv es wer e observ ed in wild-type and m utant str ains .T he results indicate that the in vitro growth of m utant str ain was almost similar to the wild-type counter part (Fig. 2 ).

msrP strain shows very mild hypersensitivity to oxidants (HOCl and ChT)
Next, we determined the effect of deletion of the msrP gene on survival of S. Typhimurium against HOCl and ChT.In comparison to the wild-type, the mutant strain was slightly (7.7 folds) ( P = 0.0612), but not significantly sensitive to 100 μM HOCl (Fig. 3 A).Mutant strain sho w ed almost similar sensitivity to 100-400 μM ChT as sho wn b y wild type (Fig. 3 B).Following incubation with 500 μM concentration of ChT, we recov er ed fe w colonies in S .Typhim urium onl y, indicating significant sensitivity of the mutant strain ( P < 0.0001) only at this concentration.

msrP strain shows analogous levels of protein carbon yla tion under oxidati v e stress
Carbon yls ar e consider ed as stable markers of protein oxidation (Dalle-Donne et al. 2003 ) and can be exploited to assess the le v els of protein oxidation as well as relative cellular susceptibility to oxidants.We quantified the total protein carbonyl levels of the whole proteome following exposure of S .Typhimurium and msrP strains to HOCl.Following HOCl exposure, msrP strain sho w ed almost similar le v els (1.08-folds) of protein carbonyls as that of S .Typhimurium (Fig. 4 ).

msrP strain exhibited hypersensitivity to neutrophils
T he reco vered numbers of wild-type and m utant str ains following incubation with neutrophils were (CFUs/ml in hundreds, mean ± SD) 508.33 ± 71.39 and 295 ± 42.30, respectiv el y.As compar ed to the wild-type, mutant strain was 1.72-folds ( P < 0.001) more susceptible to neutrophils (Fig. 5 ).

The fitness of msrP strain is not compromised in mice spleen and li v er
A se parate stud y demonstrated that the fitness of msrA , but not of msrB strain of S. Typhimurium is compromised in mice (Denkel et al. 2011 ).Ho w e v er, the contribution of msrP gene on colonization in mice is not known.
We e v aluated the fitness of msrP strain in comparison with S .Typhimurium, by inoculating a mixture of both S .Typhimurium and msrP strains in the mice .T he CI v alues ar e depicted in Table 2 .Following 3-and 5-days post-infection, we r ecov er ed slightl y higher numbers of wild type than that of mutant strain from spleen and liver.

Discussion
Intracellular pathogens like S .Typhimurium constantly encounter oxidants during colonization in the host (Noster et al.Periplasm is a narrow space located between the outer and inner cell membrane it constitutes about 10%-20% of cell volume and is the home for about 367 proteins in E. coli (Weiner and Li 2008 ).On the other hand, periplasmic proteins, due to their localization, are the prime targets of host-generated oxidants .T he oxidizing environment of periplasm further augments the oxidativ e dama ge to these pr oteins .T her efor e , bacterial pathogens ha ve e volv ed mec hanism(s), whic h can pr otect periplasmic pr oteins from o xidati ve damage.Enzymes lik e DsbA catalyze the formation of disulfide bridges in periplasmic proteins and promote their stability (Depuydt et al. 2011 ).
Very r ecentl y a unique periplasmic Msr (MsrP) system has been identified (Gennaris et al. 2015 ).Ho w e v er, the contribution of MsrP in protection of the bacterial pathogens against o xidati ve stress and virulence has not been e v aluated.MsrP along with MsrQ can r educe both fr ee and pr otein-bound as well as R-and S-stereoisomers of Met-SO (Gennaris et al. 2015, Andrieu et al. 2020 ) in E. coli as well as in S. Typhimurium.Salmonella Met auxotroph mutant lacking all Msrs ( metA 4msr cyto msrP) failed to grow on a media containing Met-SO as the sole source of methionine, suggesting that S .Typhimurium encodes only 5 Msrs (Andrieu et al. 2020 ).These findings lead us to hypothesize that msrP strain might be highly susceptible to oxidative stress.
First, we have generated msrP gene deletion strain in S .Typhim urium.Subsequentl y, we hav e assessed the contribution of msrP in resisting o xidati ve stress in vitro and survival of S. Typhimurium in vivo .Whenever there are nutritional or environmental changes, bacteria adapt itself to new microenvironment and also upregulate the expression of genes required for growth during the lag phase (Vermeersch et al. 2019 ).Adapted cells then enter into the exponential phase, where cells divide at a constant r ate (Nav arr o Llor ens et al. 2010 ).Ho w e v er, steady gr owth and exhaustion of nutrients lead to a stationary phase where bacteria encounter ROS generated by aer obic r espir ation (Dukan and Nyström 1998 ).Indeed, msrA and msrB genes were found to be upregulated during stationary phase of growth in S .Typhimurium (Rolfe et al. 2012 ).First, we analysed the effect of deletion of msrP on the in vitro growth of S. Typhimurium.We observed that the growth of msrP strain was almost similar to that of S. Typhimurium (Fig. 2 ).This indicated that deletion of msrP doesn't have an effect on in vitro growth of S. Typhimurium.
Neutrophils and, to a lesser extent, macr opha ges contain m yelopero xidase (MPO), the enzyme that catalyzes formation of hypohalous acids from H 2 O 2 and halides (Winterbourn and Kettle 2013 ).Due to the abundance of Cl − ions, the major product of the abov e r eaction is HOCl (Ulfig and Leichert 2021 ).HOCl is a k e y po w erful oxidant generated by host imm une r esponse (Klebanoff et al. 2013 ).Although H 2 O 2 is more stable than HOCl, it is less reactive and can diffuse out of phagosomes faster (3 × 10 −3 cm s −1 ), leading to collateral tissue damage.Due to its confined action inside phagosome, shorter half-life, and lesser diffusion rate, the production of HOCl is advantageous for host (Schürmann et al. 2017 ).Among the amino acids, cysteine and methionine are highly susceptible to oxidation by HOCl produced via the r espir atory burst.Met r esidues wer e observ ed to hav e the highest r eactivity to HOCl (Hawkins et al. 2003 ).
Mutants in cytoplasmic msr s of se v er al bacterial pathogens lik e, M. tuber culosis, E. coli , and P .aeruginosa sho w ed hypersensitivity to bleach stress (Lee et al. 2009, Rosen et al. 2009, Romsang et al. 2013 ).Inter estingl y, S. Typhim urium m utants lac king msrA or msrAmsrC (both genes together) genes were found to be hypersusceptible to HOCl (Trivedi et al. 2015, Nair et al. 2021 ).
In non-typeable H. influenzae (NTHi), MsrAB is localized in periplasm and is r equir ed for the survival of this bacterium a gainst bleac h str ess (Nasr een et al. 2020 ).Similarl y, a m utant str ain lac king msrP gene in E. coli was found to be moder atel y susceptible to HOCl (Gennaris et al. 2015 ).On the other hand, the expression of msrP in E. coli and msrAB in NTHi was induced following HOCl exposure (Gennaris et al. 2015, Nasreen et al. 2020 ).
A new study has found that MsrP does not hav e m uc h impact on the survival of S. Typhimurium on exposure to HOCl in growing conditions (Andrieu et al. 2023 ).
In the current study, we also observed that msrP strain sho w ed only mild sensitivity to HOCl as compared to that of wild-type strain of S. Typhimurium in non-growing conditions (Fig. 3 A).
N -c hlor otaurine is a long-acting endogenous oxidizing agent pr oduced mainl y by activ ated gr anuloc ytes and monoc ytes (Eitzinger et al. 2012 ).During an o xidati v e burst, HOCl r eacts with different amine groups and forms N -chloramines (Nagl et al. 2000 ).Within neutr ophil pha gosome, about 10%-50% of the HOCl r eacts with fr ee amines and r esults in gener ation of c hlor amines (Nagl et al. 2000 ).Since taurine accounts to more than half of the total amino acid pool in the neutr ophils, N -c hlor otaurine is the main product formed and also has more stability compared to other chloramines formed from α-amino acids (Gottardi and Nagl 2010 ).Although N -chlorotaurine is a poor oxidizing agent as compared to other N-Cl compounds, it acts as a vehicle for the strong oxidizing agent like HOCl during the oxidation burst, thus prolonging the oxidizing effect on pathogens (Anich et al. 2021 ).In a recent study, it has been shown that msrP strain of S. Typhimurium shows poor survival on exposure to N -chlorotaurine in growing conditions .T he same study established that N -c hlor otaurine induces expression of MsrP in S. Typhimurium, elucidating the necessity of this periplasmic reductase during envelope stress (Andrieu et al. 2023 ).Similarly, we assessed the effects of deletion of msrP gene on survival of S. Typhim urium a gainst ChT under non-gr owing condition.ChT is a structural analogue of N -chlorotaurine and preferentially oxidizes Met residues (Mahawar et al. 2011 ).Following incubation with ChT, mutant strain sho w ed mild susceptibility as compared to that of wild-type strain of S. Typhimurium (Fig. 3 B).
As discussed earlier, since proteins are the major target of oxidativ e dama ge and like wise man y amino acid r esidues also undergo o xidati ve modifications (Hawkins and Davies 2019 ).One such modification is carbonylation.Carbonylation is the formation of aldehyde , ketone , or lactam in amino acid residues (Fedor ov a et al. 2014 ).Since carbonyl groups are introduced very earl y during pr otein oxidation and ar e r elativ el y stable as compared to other modifications, they can be exploited to estimate extent of o xidati v e dama ge in pr oteins (Dalle-Donne et al. 2003 ).Mice lacking msr genes sho w ed high levels of protein carbonyls in various tissues following o xidati ve stress (Moskovitz et al. 2001 , Moskovitz andStadtman 2003 ).Similarly, msr inactivated or deletion mutants of pathogens like H. pylori and S .Typhimurium sho w ed incr eased pr otein carbon ylations (Alam uri andMaier 2004 , Denkel et al. 2011 ).Next, we have assessed the total protein carbonyl levels in different strains of S .Typhimurium.Following exposure to HOCl, almost identical levels of protein carbon yls wer e observ ed in m utant str ain when compar ed to that of the wild-type strain (Fig. 4 ).
Neutrophils play a very important role in host defense against Salmonella infection (Brinkmann et al. 2004 ) and are the k e y cells involved in spread of S. Typhimurium.Neutrophils use various mechanisms to kill invading bacteria (Geddes et al. 2007 ).These mechanisms include, formation of neutrophil extracellular tr a ps (NETs), secr eting cytokines, r eleasing pr oteases, and gener ation of ROS (Lie w and Kubes 2019 ).Due to the presence of Nox system (source of superoxide ion) and MPO system (source of HOCl), neutr ophils pr oduce copious amounts of ROS and RCS (Segal 2005 , Davies and Hawkins 2020 ).Indeed, MPO-deficient mice w ere sho wn to be hypersusceptible to Salmonella infection (Burton et al. 2014 ).Depletion of neutrophils using anti-RB6-8C5 antibodies resulted in enhanced Salmonella infection in mice (Vassiloyanakopoulos et al. 1998 ).Salmonella deploys various strategies to survive inside the neutrophils.One of them is a repair of its oxidized proteins by Msrs (Cheminay et al. 2004, Westerman et al. 2021 ).Indeed, S .Typhim urium m utant lac king msrA w as sho wn to have poor survival inside neutrophils (Trivedi et al. 2015 ).
To assess the degree of importance of periplasmic Msr in the survival of S .Typhimurium inside neutrophils, we performed the neutrophil sensitivity assa ys .Curiously, msrP mutant strain ( P < 0.001) was found to be highly sensitive to neutrophils in comparison to S. Typhimurium (Fig. 5 ).
Few studies used msr gene deletion strains of S .Typhimurium to elaborate on the importance of Msrs in in vivo colonization in mice (Denkel et al. 2011, Denkel et al. 2013 ).The fitness of msrA and msrC strains but not of msrB strain has been shown to be compromised in mice (Denkel et al. 2011 ).Studies pertaining to importance of periplasmic msr in in vivo survival are scarce.A mutant lacking cj0379c gene (periplasmic msr) in C .jejuni was shown to have defective colonization in poultry (Hitchcock et al. 2010 ).Inter estingl y, our r esults suggest that the msrP plays only a minor role in mice colonization (Table 2 ).
In E. coli , the enhanced expression of msrP was observed under bleach stress.Further, MsrP was found to protect the activity of periplasmic c ha per ones like SurA (Gennaris et al. 2015 ).Howe v er, our r esults suggest that the role of MsrP might be secondary to cytoplasmic Msrs in maintaining the cell viability under stress conditions .F ew studies suggested the possibility of r elativ el y high resistance of periplasmic proteins to o xidati ve damage and aggregation following o xidati ve and chemical stress.A study reported that when periplasmic proteins of E. coli were subjected to various stresses (heat stress, 60% ethanol, 0.5 M HCl, or 5 mM CuSO 4 ), they tend to stay soluble and a ggr egate less in comparison to that of non-periplasmic proteins (Liu et al. 2004 ).Another study observ ed that exposur e of E. coli to HOCl r esulted in the oxidation of Met residues in both periplasmic and cytoplasmic proteins.Howe v er, the study sho w ed that the bacterial death is linearl y pr oportional to Met oxidation in periplasmic pr oteins, onl y after 30%-40% of Met in periplasmic proteins were oxidized.In contrast to that, amount of Met oxidation in cytoplasmic and inner membr ane pr oteins is al ways linearl y pr oportional to bacterial killing, with complete bacterial death e v en at 10%-35% met oxidation in cytoplasmic proteins (Rosen et al. 2009 ).
Man y Gr am-negativ e bacteria hav e e volv ed to r estrict the entry of harmful substances by altering their membrane permeability (Martinez et al. 2001 ).Indeed, it was reported that in S .Typhimurium, permeability to H 2 O 2 is highly modulated by two outer membrane proteins (OMPs)-OmpA and OmpC.Interestingl y, OmpA was r eported to hav e a periplasmic domain with two specific cysteine residues whose oxidation status determines the opening and closing of this porin (van der Heijden et al. 2016 ).Ho w e v er, similar mec hanisms to alter membrane permeability to HOCl and n-c hlor otaurine in Gr am-negativ e bacteria as a possible way to deter oxidation of membr ane pr oteins need to be assessed.
In summary, our study suggests that periplasmic Msr plays a r elativ el y insignificant r ole in combating o xidati v e str ess and virulence of S .Typhimurium.Our findings indicate that MsrP could act as a secondary line of defence against bleach stress by indir ectl y quenc hing the incoming HOCl and c hlor amines fr om oxidizing the more susceptible cytoplasmic proteins.

Figure 1 .
Figure 1.Construction of msrP gene deletion ( msrP ) strains in S .Typhim urium.Agar ose gel analysis of different steps for msrP mutant construction.The msrP gene was deleted as described in materials and methods and confirmed by PCR.Various templates used for PCR r eactions ar e labeled on the top of v arious lanes.

Figure 2 .
Figure 2. msrP strain does not show growth defect in various in vitro media.The WT and msrP strains of S. Typhimurium were cultured in LB (2A) or M9 minimal medium (2B) .Aliquots were withdrawn at various times post inoculation and absorbance were recorded at 600 nm.Data ar e pr esented as mean ± S. D. ( n = 3).

Table 2 .Figure 3 .
Figure 3. Oxidant susceptibility assays.S .Typhimurium and msrP str ains wer e gr own in LB br oth, pelleted, suspended in PBS, and exposed to indicated concentrations of HOCl (3A) or ChT (3B) for 30 min.Excess oxidants were then quenched by addition of l -methionine .T he cultures were then serially diluted and plated on HE agar plates.Following overnight incubation, the colonies were counted and expressed as log 10 CFU per ml.Data ar e pr esented as mean ± SE ( n = 4) for HOCl; ( n = 5) for ChT.

Figure 4 .
Figure 4. Estimation of total protein carbonyl contents of whole proteome in various strains of S. Typhimurium.Cell free lysates of 0 and 3 mM HOCl-exposed cultures were derivatized with 10 mM 2, 4-DNPH.Pr otein carbon yl le v els wer e determined as described in the section 'Materials and Methods'.The data are presented as mean ± SE ( n = 3).

Figure 5 .
Figure 5. Neutrophil sensitivity assays.Different strains of S .Typhim urium wer e tr eated with neutr ophils at a r atio of 10:1 (bacteria: cells) for 60 min.Following incubation, the mix was centrifuged and the pellet was treated with 0.1% Triton-X 100.The lysates were then serially diluted and plated on HE agar.CFUs were counted following overnight incubation of the plates .T he data are presented as mean ± SD ( n = 6) and the experiment was repeated twice .T he data were analysed by paired t -test.* * P ≤ 0.001.